Hormones that bind to cell surface receptors trigger a cascade of biochemical events which result in second messenger generation and cellular activation. The membrane components involved in the receptor-regulated adenylate cyclase system, receptors, G proteins and cyclase, have been purified and functionally reconstituted in vitro. However, the actions of over 25 hormones, transmitters and growth factors are known to involve a separate transduction mechanism of receptor-regulated phosphoinositide hydrolysis and the generation of lipid-derived second messengers. Membrane components involved in this transmembrane signalling system, G proteins and phospholipase C (PL C), remain to be identified and characterized. Thyrotropin-releasing hormone (TRH) is a well-studied representative of this class of Ca2+-mobilizing hormones. Our previous studies demonstrated the GTP-dependent stimulation of PL C activity by TRH in GH3 cell plasma membranes. TRH stimulation was shown to be mediated through a novel pertussis toxin-, cholera toxin-insensitive G protein (Gp). We have developed methods to solubilize and separate GH3 cell membrane Gp and PL C. In addition, we have reconstituted functional Gp-PL C coupling in artificial liposomes using the separated components. With these techniques, it is now possible to purify and characterize the components involved in the transduction system utilized by TRH. Our specific aims for this project period are: 1. To purify and characterize the Gp that mediates receptor activation of PL C; and 2. To purify and characterize the membrane PL C that is regulated by the receptor and Gp. In additional studies, the regulation of Gp and PL C by the TRH receptor and by protein kinase C will be studied utilizing reconstituted systems. Hence, the additional specific aim will be: 3. To reconstitute TRH receptor-Gp-PL C coupling in artificial liposomes and to elucidate the mechanism for reduced Gp-PL C coupling in phorbol ester-treated GH3 cells. The complete characterization of components involved in TRH-initiated signal transduction will significantly contribute to a broader understanding of the mechanism by which Ca2+-mobilizing hormones trigger cellular responses mediated through cell surface receptors.